Abstract/Summary In this proposal I will extend our initial examinations with wild type NOD2 to disease-associated mutant NOD2 proteins. We have taken advantage of distinct mutations in NOD2 to dissect the molecular mechanisms that cause NOD2-dependent pathologies. Both gain- and loss-of-function mutations of NOD2 are associated with human diseases. We propose to: 1) characterize the nucleotide-binding properties of disease-associated NOD2 proteins, 2) define the role of the nucleotide binding cycle to ascertain how it contributes to NOD2 biological function, and 3) establish the role of nucleotide binding or hydrolysis on the biological activity of disease-associated mutations of NOD2 that are linked to Blau syndrome (early onset sarcoidosis) and Crohn's disease in vivo. We believe these studies will shed light on both the mechanism of disease pathogenesis and mucosal inflammation. Studies aimed at evaluating the nucleotide binding properties are crucial for rationale design of inhibitors. Understanding the nucleotide binding properties of NOD2 may ultimately lead to the development of pharmacologic agents that can modify signaling by NOD2, which would have clinical application in systemic and intestinal inflammatory conditions. !